In the adrenal medulla and sympathetic neuron intracellular vesicles called chromaffin granules are involved with the biosynthesis, storage, and secretion of catecholamines. The membranes surrounding these chromaffin granules uniquely contain a single b-type cytochrome or which neither the electron donor nor acceptor is known. The membranes also contain an NADH oxidoreductase activity which is inhibited by sulfhydryl-reactive reagents but which has not been further characterized. The cytochrome and NADH oxidoreductase will be isolated and purified from the chromaffin granule membrane using detergent extraction, ion exchange, hydrophobic, gel filtration, and affinity chromatography. These enzymes will be characterized in terms of molecular weight, isoelectric point, amino acid composition and amino terminus using standard standard techniques. The presence of discrete catalytic and membrane binding domains will be investigated using limited proteolytic digestion. The catalytic site orientation of the enzymes relative to the interior or exterior of the granule membranes will be determined using permeant and impearmeant labeled amino, carboxyl, and sulfhydryl reagents. The binding of these enzymes to artificial lipid membranes will be characterized in terms of protection from proteolytic cleavage. Electron transfer between the purified NADH oxidoreductase and cytochrome will be investigated with the enzymes reconstituted in phospholipid vesicular membranes containing lecithin with varying proportions of lysolecithin, cholesterol, and ethanolamine plasmalogen. A search for the function of the electron transfer system in the chromaffin granule membrane will begin with attempts to demonstrate electron transfer from the purified cytochrome to purified dopamine-beta-hydroxylase which is normally bound to the interior surface of the granule membrane. As a preliminary to this latter work the binding of dopamine-beta-hydroxylase to artificial phospholipid membranes will also be attempted.